Direct capture and selective elution of a secreted polyglutamate-tagged nanobody using bare magnetic nanoparticles.

BACKGROUND: The secretion and direct capture of proteins from the extracellular medium is a promising approach for purification, thus enabling integrated bioprocesses. MAJOR RESULTS: We demonstrate the secretion of a nanobody (VHH) to the extracellular medium (EM) and its direct capture by bare, non-functionalized magnetic nanoparticles (MNPs). An ompA signal peptide for periplasmic localization, a polyglutamate-tag (E8 ) for selective MNP binding, and a factor Xa protease cleavage site were fused N-terminally to the nanobody. The extracellular production of the E8 -VHH (36 mg L-1 ) was enabled using a growth-decoupled Escherichia coli-based expression system. The direct binding of E8 -VHH to the bare magnetic nanoparticles was possible and could be drastically improved up to a yield of 88% by adding polyethylene glycol (PEG). The selectivity of the polyglutamate-tag enabled a selective elution of the E8 -VHH from the bare MNPs while raising the concentration factor (5x) and purification factor (4x) significantly. CONCLUSION: Our studies clearly show that the unique combination of a growth-decoupled E. coli secretion system, the polyglutamate affinity tag, non-functionalized magnetic nanoparticles, and affinity magnetic precipitation is an innovative and novel way to capture and concentrate nanobodies.

Selective ene-reductase immobilization to magnetic nanoparticles through a novel affinity tag.

BACKGROUND: Magnetic nanoparticles (MNPs) are becoming more important as carriers, because of their large specific surface area and easy separability. They are increasingly used in enzyme technology, diagnostics, and drug delivery. MAJOR RESULTS: For the directed and almost irreversible immobilization of proteins on MNPs, we have developed a new selective (His-Arg)4 peptide-tag, that binds fusion proteins directly from an E. coli cell lysate to non-functionalized, low-cost MNPs. Using the immobilization of an ene-reductase as an example, we could demonstrate that the fusion with this tag increases thermostability without reducing overall activity (ER w/o tag: t1/2  = 3.7 h, (HR)4 -ER: t1/2  = 9.9 h). Immobilization by adsorption in Tris buffer resulted in very high enzyme loads with approx. 380 mg g-1 and 67% residual activity. The immobilization on the MNPs allowed a fast concentration, buffer exchange, and reuse. While about 50% of the activity was lost after the first reuse, we were able to show that the activity did not decrease further and was stable for another nine cycles. CONCLUSION: According to our studies, our tag highly works for any kind of immobilization on MNPs and holds the potential for enzyme immobilizations as well as for drug delivery and sensors.